Our long term objective is to understand the regulation of extracellular matrix (ECM) production by human fibroblasts and its dysregulation in fibrotic diseases such as scleroderma (systemic sclerosis, SSc). Organ fibrosis, a main pathologic manifestation of SSc, is the result of excessive deposition of ECM. Numerous studies have established a central role for transforming growth factor-beta (TGF-beta) in the fibrogenic process occurring in various organs and diseases including SSc. Rapid progress made in the past several years has led to the identification and characterization of the several key components of the TGF-beta signaling pathway. However, it is still not fully understood how TGF-beta signaling is integrated with other cellular signaling pathways to achieve diverse tissue-specific responses. Furthermore, the nature of the specific alterations of TGF-beta signaling that contribute to the fibrogenic process in SSc is not yet clear. We have made a novel observation that in human fibroblasts TGF-beta regulates levels and activities of the enzymes involved in regulation of lipid metabolism. Specifically, TGF-beta upregulates the levels and activity of the sphingosine kinase (SPHK) and down regulates activity of the sphingosine phosphate phosphatase (S1Pase). Furthermore, our preliminary data using ectopically expressed SPHK suggest that SPHK inhibits TGF-beta stimulation of collagen production. Significantly, basal and TGF-beta-induced activity of SPHK is lower in SSc skin and lung fibroblasts. Based on these observations we hypothesize that in normal fibroblasts TGF-beta activates SPHK and induces the levels of sphingosine 1-phosphate (S1P), which in turn provides a counter regulatory signal (negative autofeedback loop) affecting duration and/or intensity of TGF-beta stimulation of collagen production, and that this signaling pathway is defective in SSc fibroblasts. We propose three specific aims to test these hypotheses and to start unraveling the molecular mechanisms of the cross-talk between sphingolipids and the TGF-beta. In Specific Aim 1 we will determine whether the sphingolipid pathway is dysregulated in SSc fibroblasts by examining the levels and activities of the key lipid enzymes and determining the intracellular concentrations of ceramide, sphingosine, and sphingosine 1-phosphate (S1P) in SSc and healthy dermal fibroblasts at the basal level and after TGF-beta stimulation. In Specific Aim 2 we will determine the mechanism by which the SPHK/S 1P pathway regulates ECM production in response to TGF-beta signaling. in Specific Aim 3 we will determine the role of sphingosine phosphate phosphatase (S1Pase) and sphingosine phosphate lyasc (S1P lyase), two important enzymes involved in regulation of the cellular levels of S 1P, in TGF-beta signaling and collagen metabolism in SSc and healthy fibroblasts.